The extrinsic pathway of blood coagulation is initiated when FVIIa circulating in plasma binds to the integral-membrane protein, tissue factor (TF). The role of TF in blood coagulation has been extensively studied. The involvement of FVIIa as a proteolytic enzyme in the blood coagulation cascade is believed to be confined to the extracellular leaflet of TF expressing cells. An intracellular activity of FVIIa was first implied when the sequence of TF showed homology to the cytokine/interferon- or heamatopoietic receptor superfamily. The subclass I of the heamotopoietic receptor family includes receptors for growth hormone, prolactin, interleukins 1 to 7, granulocyte-macrophage colony stimulating factors, erythropoitin and thrombopoitin. Subclass II includes TF and receptors for interferon a and b.
The resemblance of TF to this class of receptors was further substantiated with the appearance of the crystal structure. Characteristic of this class of cytokine receptors that includes receptors for interferon b and g and IL-10 is that their activation lead to rapid tyrosine phosphorylation of the receptors themselves, as well as a subset of intracellular proteins. Within minutes after the initial tyrosine phosphorylation an array of mitogen-activated (Ser/Thr) kinases (MAPK) is activated. These kinases are arranged in several parallel signalling pathways. Thorough studies of the putative intracellular signalling capacity of FVIIa have shown that it induce mobilisation of intracellular free calcium (Ca2+) in the human bladder carcinoma cell line, J82, which constitutively express TF and in umbelical vein endothelial cells which were pre-treated with interleukin-1 to express TF, but have failed to show any cytokine-like activation of intracellular tyrosine kinases. In conclusion FVIIa is believed, in a TF dependent manner, to induce mobilisation of intracellular Ca2 + through activation of phospholipase C. The mechanism by which FVIIa activates phospholipase c is not known, but tyrosine kinase activation has specifically been ruled out.
Recent reports from a number of laboratories indicate that TF may influence an array of important biological functions other than coagulation., such as angiogenesis, embryo vascularization and tumor metastasis. At present, however, it is unclear how TF contributes to these biological processes. The extracellular domain of TF consists of two fibronectin-type III-like modules, as in the typical class II cytokine receptor extracellular domain, raising the possibility that TF may play a role in signal transduction, the primary function of cytokine receptor. However, TF has a very short cytoplasmic domain (only 21 amino acid residues in length) and lacks membrane-proximal motifs that mediate binding of the non-receptor Janus kinases (Jaks) that are essential for cytokine receptor signaling. Nonetheless, several biochemical findings suggest a signal transduction function for TF. Analysis of the human TF protein sequence revealed a putative phosphorylation site in the cytoplasmic domain, which is conserved in mouse, rat and rabbit TF. Specific serine residues in the cytoplasmic tail of TF are phosphorylated in cells following stimulation with protein kinase C activator. The human TF cytoplasmic tail is phosphorylated in vitro at multiple sites when incubated with lysates of U87-MG cells. A potential role for the TF cytoplasmic domain in signal transduction is also indicated in studies that showed prometastatic function of TF is critically dependent on the TF cytoplasmic domain. Further, TF cytoplasmic domain is shown to interact with actin-binding protein 280 (ABP-280) and supports cell adhesion and migration through recruitment of ABP-280 to TF-mediated adhesion contacts.
However, TF has also been shown to participate certain types of cell signaling by serving as a cofactor for its physiological ligand FVIIa in an extracellular signaling by a proteolytic mechanism. For example, binding of FVIIa to cell surface TF is shown to induce intracellular Ca2 + oscillations in a number of TF expressing cells, transient phosphorylation of tyrosine in monocytes, activation of MAP kinase, alteration in gene expression in fibroblasts and enhanced expression of urokinase receptor in tumor cells. Catalytically inactive FVIIa (FVIIai) fails to induce many of the above signaling responses, from Ca2 + oscillations to MAP kinase activation and gene reduction, and it appears that the catalytic activity of FVIIa may be required for at least some TF-FVIIa-mediated signal transduction. At present, not much is known about signaling pathway(s) that are induced by proteolytically active FVIIa and how the signals generated by FVIIa could contribute to angiogenesis and tumor metastasis.
To study temporal program of transcription that underlies the FVIIa-induced response, in the present study, we have examined the response of human fibroblasts to FVIIa using a cDNA microarray. The data revealed that the cellular expression of several genes was detectably altered in fibroblasts upon exposure of to FVIIa. One such gene is Cyr61, a growth factor-inducible intermediate early gene, whose product is shown to promote cell adhesion, augment growth factor-induced DNA synthesis and stimulate cell migration in fibroblasts and endothelial cells.